US11008547B2 - Passive replacement of media - Google Patents

Passive replacement of media Download PDF

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US11008547B2
US11008547B2 US14/668,659 US201514668659A US11008547B2 US 11008547 B2 US11008547 B2 US 11008547B2 US 201514668659 A US201514668659 A US 201514668659A US 11008547 B2 US11008547 B2 US 11008547B2
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media
cells
bag
inlet
bioreactor
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US20150275170A1 (en
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Brian J. NANKERVIS
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Terumo BCT Inc
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0602Vertebrate cells
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/10Hollow fibers or tubes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00Means for introduction, extraction or recirculation of materials, e.g. pumps
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/16Hollow fibers
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/26Conditioning fluids entering or exiting the reaction vessel
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08Chemical, biochemical or biological means, e.g. plasma jet, co-culture
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48Automatic or computerized control

Definitions

  • Cell Expansion Systems are used to expand and differentiate cells.
  • Cell expansion systems may be used to expand, e.g., grow, stem cells, such as mesenchymal stem cells, human mesenchymal stem cells, etc.
  • Cell expansion systems may also expand other types of cells, such as bone marrow cells, for example.
  • Stem cells which are expanded from donor cells may be used to repair or replace damaged or defective tissues and have broad clinical applications for a wide range of diseases.
  • Cells, of both adherent and non-adherent type may be grown in a bioreactor in a cell expansion system.
  • Embodiments of the present disclosure generally relate to using the passive replacement of media in a cell expansion system to conserve media and provide an environment conducive to encouraging cell growth.
  • the expansion of cells such as human mesenchymal stem cells, for example, uses external chemical signaling between the cells to initiate cell expansion by inhibiting lag phase signaling pathways internal to the cells.
  • the expansion of other types of cells such as Chinese hamster ovary (CHO) cells, for example, may be particularly sensitive to chemical signaling between the cells, according to embodiments.
  • CHO cells secrete cholecystokinin (CCK), a regulatory hormone responsible in part for cell culture maintenance and proliferation via chemical signaling.
  • CCK may be small enough to pass through the microporous membrane of a hollow fiber bioreactor.
  • aspects of particular embodiments provide for passively replacing media by interrupting protocol procedures being executed and replacing a waste or outlet bag(s) used with the cell expansion system with a media bag(s).
  • a bag containing base media may be attached to a waste line of the cell expansion system, in which such configuration allows base media to be added to the system at the rate of evaporation during conditions of no active inlet fluid flow.
  • fluid may be passively replaced by interrupting protocol procedures being executed and allowing any fluid in the waste or outlet bag (assuming no constituents toxic to cell growth are present in the waste or outlet bag) to be passively added to the system at the rate of evaporation during conditions of no active inlet fluid flow.
  • the passive addition of fluid avoids adding an excess amount of fluid, in which an excess amount of fluid may dilute the chemical signaling used to initiate cell expansion. Further, media constituents themselves may ultimately be conserved, resulting in increased system efficiencies and a savings of resources.
  • Embodiments of the present disclosure further relate to enhancing chemical signaling by adding a molecule(s), e.g. cell-signaling protein molecules, such as cytokines, according to embodiments, to the expanding cell population in a bioreactor.
  • a molecule(s) e.g. cell-signaling protein molecules, such as cytokines
  • cytokines, or other type of cell-signaling protein molecules may be added to the bioreactor by, for example, welding a tubing line or other material connected to a cytokine source, or pre-filled with cytokines or other desired constituents, to a sampling coil or sample coil of the cell expansion system. The cytokines may thus be added to the bioreactor at the sample coil.
  • cytokines tend to degrade quickly over time or with exposure to ultra-violet (UV) light, in which such degradation may be minimized by adding cytokines closer to the expanding cell population, e.g., at the sample coil of the bioreactor itself which is isolated from UV light sources.
  • UV ultra-violet
  • the cytokines in the bioreactor may thus be maintained at a certain level while conserving resources.
  • each of the expressions “at least one of A, B and C,” “at least one of A, B, or C,” “one or more of A, B, and C,” “one or more of A, B, or C” and “A, B, and/or C” may mean A alone, B alone, C alone, A and B together, A and C together, B and C together, or A, B and C together.
  • FIG. 1 depicts a perspective view of a hollow fiber bioreactor, in accordance with embodiments of the present disclosure.
  • FIG. 2 illustrates a perspective view of a cell expansion system with a premounted fluid conveyance device, in accordance with embodiments of the present disclosure.
  • FIG. 3 depicts a perspective view of a housing of a cell expansion system, in accordance with embodiments of the present disclosure.
  • FIG. 4 illustrates a perspective view of a premounted fluid conveyance device, in accordance with embodiments of the present disclosure
  • FIG. 5 depicts a schematic of a cell expansion system, in accordance with an embodiment of the present disclosure.
  • FIG. 6 illustrates a schematic of another embodiment of a cell expansion system.
  • FIG. 7 depicts the cell expansion system embodiment of FIG. 6 with a waste bag replaced by a media bag, in accordance with embodiments of the present disclosure.
  • FIG. 8 illustrates the cell expansion system embodiment of FIG. 5 with a waste bag replaced by a media bag, in accordance with embodiments of the present disclosure.
  • FIG. 9 depicts the cell expansion system embodiment of FIG. 6 with a molecule source included as part of the premounted fluid conveyance device, in accordance with embodiments of the present disclosure.
  • FIG. 10 illustrates a flow diagram depicting the operational characteristics of a process for passively replacing media in a cell expansion system, in accordance with embodiments of the present disclosure.
  • FIG. 11 depicts a flow diagram illustrating the operational characteristics of a process for adding a molecule from a molecule source implemented as part of the cell expansion system itself, in accordance with embodiments of the present disclosure.
  • FIG. 12 illustrates a flow diagram depicting the operational characteristics of another embodiment of a process for passively replacing media in a cell expansion system.
  • FIG. 13 depicts an example processing system of a cell expansion system upon which embodiments of the present disclosure may be implemented.
  • Embodiments of the present disclosure are generally directed to systems and methods for passively replacing media in a cell expansion system. Passive replacement of media may be accomplished by interrupting one or more protocol procedures being executed with respect to the system, e.g., cell loading, cell feeding, etc., and replacing a waste or outlet bag(s) used with the system with a media bag(s).
  • active inlet fluid flow to the system may be halted to reduce or prevent the dilution of chemical signaling used to inhibit the internal signaling pathways that keep a cell population in the lag phase in a bioreactor of the closed system. Reducing or preventing such dilution may thus reduce the lag phase of cell growth. More efficient and increased cell expansion may therefore occur, in which a greater number of cells may be expanded in a shorter amount of time, according to embodiments of the present disclosure.
  • Dilution of chemical signaling may occur where an inlet fluid flow into a cell expansion system overcompensates for the evaporation of fluid from the system.
  • an oxygenator or gas transfer module may be used in a closed cell expansion system to maintain the media in fibers in the bioreactor with a desired gas concentration, e.g., 5% CO 2 , 20% O 2 , 75% N 2 .
  • evaporation in the gas transfer module may occur at 14 mL/day. Without any inlet flow, such evaporation could result in either a build-up of air in the system or a back-flow of fluid from the waste or outlet line in embodiments where the waste line is the only source of fluid for the system which is not occluded by a pump, for example.
  • the inlet flow rate into the cell expansion system may have a minimum flow rate.
  • the inlet flow rate may be set at a minimum rate of 0.1 mL/min or 144 mL/day.
  • the fluid lost due to evaporation may be overcompensated for by a rate of 130 mL/day in such embodiment.
  • Such excess 130 mL/day dilutes chemical signaling for initiating cell expansion.
  • such dilution may occur in embodiments where chemical signaling molecules are able to cross, or pass through, a hollow fiber membrane from an intracapillary to an extracapillary side.
  • adding replacement fluid to either the intracapillary or extracapillary side may result in dilution of the chemical signaling molecules by preventing or reducing them from building up by continuously adding fluid into the system.
  • communication between the chemical signaling cells may be significantly impacted such that the cells may be unable to expand or even survive.
  • Such dilution may have a particularly significant impact with respect to some cell types as compared to others.
  • reducing or preventing the dilution of chemical signaling molecules may have a significant impact on the expansion of Chinese hamster ovary (CHO) cells, according to embodiments.
  • the active inlet fluid flow to the system may be halted to prevent or minimize the dilution of chemical signaling used to inhibit the signaling pathways that maintain the cell population in a bioreactor in the lag phase.
  • Such active inlet fluid flow may be halted, for example, by interrupting, or stopping, system mechanisms from operating according to the protocol(s) being executed.
  • active inlet fluid flow may therefore be stopped while using a passive replacement of media and, therefore, not result in a build-up of air or back-flow of waste.
  • fluid e.g., base media
  • fluid may be added to the system at a rate equal to the rate of evaporation from the system, e.g., such as the rate of evaporation from a gas transfer module, through the use of one or more media bags used to replace one or more waste or outlet bags normally used with the system.
  • the active inlet fluid flow may therefore be stopped while media from the replacement, or substitute, media bag replaces any fluid lost from the system due to evaporation.
  • Such passive addition of fluid avoids adding an excess amount of fluid, in which an excess amount of fluid may dilute the chemical signaling used to initiate cell expansion.
  • lost fluid may be replaced by adding media at about the rate of evaporation and without diluting chemical signaling used to inhibit signaling pathways that keep the cell population in the lag phase.
  • the lag phase of cell growth may therefore be significantly reduced.
  • media constituents themselves may ultimately be conserved, resulting in increased system efficiencies and a savings of resources.
  • fluid may be passively replaced by interrupting protocol procedures being executed and allowing any fluid in the waste or outlet bag (assuming no constituents toxic to cell growth are present in the waste or outlet bag) to be passively added to the system at the rate of evaporation during conditions of no active inlet fluid flow.
  • the dilution of chemical signaling may be particularly costly where the cell media includes expensive additives.
  • cell-signaling proteins e.g., cytokines
  • cytokines may be used in the bioreactor to spur cell growth. Diluting cytokines may thus result in significant costs. Accordingly, saving the excess media, e.g., 130 mL/day, may provide significant cost savings over other cell expansion processes.
  • the passive replacement of media may thus be used, according to embodiments of the present disclosure, to maintain media constituent concentrations and conserve media in general.
  • other types of replacement fluids are used in the media bag(s), such as, for example, a media bag comprising cytokines or other cell-signaling protein molecules.
  • molecules such as cell-signaling protein molecules
  • a source of such molecules may be added to the bioreactor from a source of such molecules.
  • tubing or other material connected to a molecule source such as a cytokine source
  • cytokines in the bioreactor may be replenished by such direct source of cytokines.
  • such tubing or other material comprises an additional volume added to the sampling coil.
  • such tubing or other material comprises a segment of tubing or other material used to replace a corresponding segment, or portion, of the sampling coil.
  • such tubing or other material may be pre-filled with the desired constituents, e.g., cytokines.
  • such tubing or material may be connected to a container or bag comprising such desired constituents.
  • a source of cytokines conserves the amount of cytokines used because the cytokines are not added to an IC media bag, for example, which could dilute the cytokines and use a larger amount of cytokines to achieve the same replenishment concentrations.
  • the cytokines may be added closer to the expanding cell population to minimize degradation of the cytokines. Degradation of the cytokines increases with exposure time to the media bags and UV light where they may be stored. Where cytokines are added closer to the expanding cell population, such degradation may be reduced because the cytokines reach the expanding cell population in a shorter amount of time in an environment protected from any sources of UV light.
  • Such cytokines may be passively or actively added to the bioreactor, according to embodiments, to enhance chemical signaling capabilities. For example, such passive addition of cytokines may occur where the cytokines are added to the system from a media bag used to replace a waste bag, according to an embodiment, at the rate of evaporation during conditions of no active inlet fluid flow.
  • chemical signaling may thus be controlled by the addition of cytokines at the sample coil.
  • chemical signaling may be controlled through such addition of cytokines at the sample coil coupled with the replacement of a waste bag(s) with a media bag(s).
  • dilution in the bioreactor may be significantly reduced, as discussed above.
  • Such dilution may be particularly costly where cytokines are used in the cell population expansion in the bioreactor. Preventing or reducing such dilution through the use of the media bag replacement thus may result in significant savings, according to embodiments.
  • a method provides for controlling chemical signaling in a bioreactor of a closed cell expansion system that includes a disposable tubing set(s).
  • the method may include the steps of coating the bioreactor and loading cells from a cell inlet bag into the bioreactor. For example, steps for loading cells with circulating distribution may be performed, according to an embodiment. In another embodiment, steps involving the loading of cells with uniform suspension, for example, may be performed. The cells may then be distributed across a membrane of the bioreactor by activating an intracapillary circulation pump, for example. In embodiments, after the loading and the distributing, a waste bag attached to the cell expansion system may be replaced with a media bag.
  • one or more pumps may be turned “OFF” or otherwise deactivated, according to an embodiment.
  • one or more pumps may be turned “OFF” or otherwise deactivated before replacing the outlet or waste bag.
  • the intracapillary circulation pump may be deactivated after replacing a waste or outlet bag with a media bag.
  • expanding adherent cells for example, the intracapillary circulation pump may be deactivated before replacing the waste or outlet bag with a media bag.
  • expanding non-adherent cells for example, the intracapillary circulation pump may stay activated while one or more other pumps are deactivated.
  • the media from the media bag flows through an extracapillary waste valve to the extracapillary circulation loop to replenish fluid evaporated from a gas transfer module in the extracapillary circulation loop.
  • the method further includes deactivating an intracapillary inlet pump, deactivating an extracapillary inlet pump, maintaining an extracapillary circulation pump in an activated state, and maintaining the extracapillary waste valve in an open position.
  • the cells include adherent cells
  • the method may include the additional steps of enabling the adherent cells to attach to the bioreactor membrane and maintaining flow on an extracapillary circulation loop by maintaining an extracapillary circulation pump in an activated state.
  • the adherent cells are allowed to attach to the bioreactor membrane for a period of time, e.g., a first period of time, of about eighteen (18) hours to about twenty-four (24) hours.
  • the cells include non-adherent or suspension cells, such as, for example, CHO cells.
  • the method may further include feeding the cells in the bioreactor of the closed cell expansion system while maintaining the media bag in replacement of the waste bag and while reducing an intracapillary inlet rate.
  • feeding may include activating the intracapillary circulation pump.
  • the feeding of the cells may be stopped after a second period of time of about forty-five (45) hours to about fifty (50) hours of feeding.
  • the feeding may be stopped after a second period of time of about forty-eight (48) hours of feeding.
  • the method further involves measuring a concentration of lactate generated from the cells and stopping the feeding of the cells when the concentration of lactate is equal to or greater than about 6 mmol/L.
  • the method includes removing the media bag, inserting the waste bag, activating the intracapillary inlet pump, activating the intracapillary circulation pump, and maintaining an extracapillary circulation pump in an activated state.
  • the intracapillary inlet pump may operate at an intracapillary inlet rate of about 0.1 mL/min, in some embodiments.
  • the intracapillary circulation pump may operate at an intracapillary circulation rate of about 20 mL/min, in some embodiments.
  • the extracapillary circulation pump may operate at an extracapillary circulation rate of about 30 mL/min, according to embodiments.
  • the method may additionally involve doubling, or otherwise increasing, according to other embodiments, the intracapillary inlet rate until a desired number of the cells are available for harvest.
  • embodiments include the additional steps of: releasing the cells from the membrane of the bioreactor, suspending the cells in the intracapillary circulation loop, and transferring the cells in suspension to a harvest bag.
  • Steps performed may be performed automatically in some embodiments, such as by a processor executing pre-programmed tasks stored in memory.
  • Replacing a waste bag with a media bag may be performed manually in some embodiments and automatically in others.
  • the automatic replacement of the waste bag may include, in embodiments, receiving, by a processor, a command to execute a task for replacing the waste bag, the task being stored in a memory.
  • a processor may send a signal to close a valve(s), for example, for the waste bag and open another valve(s) for an attached media bag.
  • a single valve, or other type of mechanism may control the flow of fluid from the waste bag or attached media bag.
  • the media bag may store base media and, in some embodiments, stores about 500 mL of base media, for example.
  • the base media may include a number of different components, including, for example, glucose to provide an energy source for cells to grow, according to an embodiment.
  • the media bag may comprise other fluids and/or constituents in accordance with embodiments of the present disclosure.
  • inventions of the method provide for additional steps, some of which include loading cell-signaling protein molecules into a sample coil of an intracapillary circulation loop and activating the intracapillary circulation pump to transfer the cell-signaling protein molecules to the bioreactor.
  • the sample coil and the intracapillary circulation loop are part of a disposable tubing set.
  • the method may further include, prior to loading cells into the bioreactor, replacing fluid on an intracapillary circulation loop and on an extracapillary circulation loop with media from an intracapillary media bag, and allowing the media from the intracapillary media bag to reach equilibrium with a gas supply.
  • Some embodiments are directed to a cell expansion system, as noted above.
  • such cell expansion system is closed, in which a closed cell expansion system comprises contents that are not directly exposed to the atmosphere.
  • Such cell expansion system may be automated.
  • cells of both adherent and non-adherent type, may be grown in a bioreactor in the cell expansion system.
  • the cell expansion system may include base media. Methods for replenishment of media are provided for cell growth occurring in a bioreactor of the closed cell expansion system.
  • the bioreactor used with such systems may be a hollow fiber bioreactor. Many types of bioreactors may be used in accordance with embodiments of the present disclosure.
  • the system may include, in embodiments, a bioreactor that further includes a first fluid flow path having at least opposing ends, a first opposing end of the first fluid flow path fluidly associated with a first port of a hollow fiber membrane and a second end of the first fluid flow path fluidly associated with a second port of the hollow fiber membrane, wherein the first fluid flow path comprises an intracapillary portion of the hollow fiber membrane.
  • the system may further include a fluid inlet path fluidly associated with the first fluid flow path, wherein the plurality of cells are introduced into the first fluid flow path through a first fluid inlet path.
  • a first pump for circulating fluid in the first fluid flow path of the bioreactor may also be included.
  • the system includes a controller, e.g., first controller, for controlling operation of the first pump.
  • the controller may be a computing system, including a processor, for example.
  • the controller may be configured, in embodiments, to control the pump to circulate a fluid at a first rate within the first fluid flow path, and, when a waste bag in the cell expansion system is replaced with a media bag, the controller stops the circulation of the fluid within the first fluid flow path after the plurality of the cells are loaded into the bioreactor.
  • a second pump for transferring intracapillary inlet fluid from an intracapillary media bag to the first fluid flow path and a controller, e.g., second controller, for controlling operation of the second pump are included.
  • the second controller in embodiments controls the second pump to transfer cells from a cell inlet bag to the first fluid flow path, and when a waste bag in the cell expansion system is replaced with a media bag, stop the transfer of the cells from the cell inlet bag after the plurality of the cells are loaded into the bioreactor.
  • Additional controllers e.g., third controller, fourth controller, fifth controller, sixth controller, etc.
  • additional pumps e.g., third pump, fourth pump, fifth pump, sixth pump, etc., may be used in accordance with embodiments of the present disclosure.
  • a media bag, a waste bag, a cell inlet bag, etc. multiple bags, e.g., a first media bag, a second media bag, a third media bag, a first waste bag, a second waste bag, a third waste bag, a first cell inlet bag, a second cell inlet bag, a third cell inlet bag, etc., or other types of containers, may be used in embodiments.
  • a single media bag, a single waste bag, a single cell inlet bag, etc. may be used.
  • the system may be controlled by, for example: a processor coupled to the cell expansion system; a display device, in communication with the processor, and operable to display data; and a memory, in communication with and readable by the processor, and containing a series of instructions.
  • the processor when the instructions are executed by the processor, the processor receives an instruction to coat the bioreactor, for example.
  • the processor may execute a series of steps to coat the bioreactor and may next receive an instruction to load cells into the bioreactor, for example.
  • the processor may execute a series of steps to load the cells from a cell inlet bag into the bioreactor.
  • the processor may receive an instruction to stop an intracapillary inlet pump, an intracapillary circulation pump, and an extracapillary inlet pump, for example.
  • the cell expansion system may be operated to allow media to flow from a media bag through an extracapillary waste valve, wherein the extracapillary waste valve is in an open position.
  • the processor may receive an instruction to pump the media in the extracapillary circulation loop to replace fluid evaporated from a gas transfer module located in the extracapillary circulation loop.
  • Cell growth chamber 100 has a longitudinal axis LA-LA and includes cell growth chamber housing 104 .
  • cell growth chamber housing 104 includes four openings or ports: IC inlet port 108 , IC outlet port 120 , EC inlet port 128 , and EC outlet port 132 .
  • fluid in a first circulation path enters cell growth chamber 100 through IC inlet port 108 at a first longitudinal end 112 of the cell growth chamber 100 , passes into and through the intracapillary side (referred to in various embodiments as the intracapillary (“IC”) side or “IC space” of a hollow fiber membrane) of a plurality of hollow fibers 116 , and out of cell growth chamber 100 through IC outlet port 120 located at a second longitudinal end 124 of the cell growth chamber 100 .
  • the fluid path between the IC inlet port 108 and the IC outlet port 120 defines the IC portion 126 of the cell growth chamber 100 .
  • Fluid in a second circulation path flows in the cell growth chamber 100 through EC inlet port 128 , comes in contact with the extracapillary side or outside (referred to as the “EC side” or “EC space” of the membrane) of the hollow fibers 116 , and exits cell growth chamber 100 via EC outlet port 132 .
  • the fluid path between the EC inlet port 128 and the EC outlet port 132 comprises the EC portion 136 of the cell growth chamber 100 . Fluid entering cell growth chamber 100 via the EC inlet port 128 may be in contact with the outside of the hollow fibers 116 .
  • Small molecules may diffuse through the hollow fibers 116 from the interior or IC space of the hollow fiber to the exterior or EC space, or from the EC space to the IC space.
  • Large molecular weight molecules, such as growth factors, are typically too large to pass through the hollow fiber membrane, and remain in the IC space of the hollow fibers 116 .
  • the media may be replaced as needed, in embodiments.
  • Media may also be circulated through an oxygenator or gas transfer module to exchange gasses as needed.
  • Cells may be contained within a first circulation path and/or a second circulation path, as described below, and may be on either the IC side and/or EC side of the membrane, according to embodiments.
  • the material used to make the hollow fiber membrane may be any biocompatible polymeric material which is capable of being made into hollow fibers.
  • One material which may be used is a synthetic polysulfone-based material, according to an embodiment of the present disclosure.
  • the surface may be modified in some way, either by coating at least the cell growth surface with a protein such as fibronectin or collagen, or by exposing the surface to radiation.
  • Gamma treating the membrane surface allows for attachment of adherent cells without additionally coating the membrane with fibronectin or the like.
  • Bioreactors made of gamma treated membranes may be reused.
  • Other coatings and/or treatments for cell attachment may be used in accordance with embodiments of the present disclosure.
  • the CES 200 includes a cell expansion machine 202 that comprises a hatch or closable door 204 for engagement with a back portion 206 of the cell expansion machine 202 .
  • An interior space 208 within the cell expansion machine 202 includes features adapted for receiving and engaging a premounted fluid conveyance assembly 210 .
  • the premounted fluid conveyance assembly 210 may be detachably-attachable to the cell expansion machine 202 to facilitate relatively quick exchange of a new or unused premounted fluid conveyance assembly 210 at a cell expansion machine 202 for a used premounted fluid conveyance assembly 210 at the same cell expansion machine 202 .
  • a single cell expansion machine 202 may be operated to grow or expand a first set of cells using a first premounted fluid conveyance assembly 210 and, thereafter, may be used to grow or expand a second set of cells using a second premounted fluid conveyance assembly 210 without needing to be sanitized between interchanging the first premounted fluid conveyance assembly 210 for the second premounted fluid conveyance assembly 210 .
  • the premounted fluid conveyance assembly includes a bioreactor 100 and an oxygenator or gas transfer module 212 .
  • Tubing guide slots are shown as 214 for receiving various media tubing connected to premounted fluid conveyance assembly 210 , according to embodiments.
  • FIG. 3 illustrates the back portion 206 of cell expansion machine 202 prior to detachably-attaching a premounted fluid conveyance assembly 210 ( FIG. 2 ), in accordance with embodiments of the present disclosure.
  • the closable door 204 (shown in FIG. 2 ) is omitted from FIG. 3 .
  • the back portion 206 of the cell expansion machine 202 includes a number of different structures for working in combination with elements of a premounted fluid conveyance assembly 210 .
  • the back portion 206 of the cell expansion machine 202 includes a plurality of peristaltic pumps for cooperating with pump loops on the premounted fluid conveyance assembly 210 , including the IC circulation pump 218 , the EC circulation pump 220 , the IC inlet pump 222 , and the EC inlet pump 224 .
  • the back portion 206 of the cell expansion machine 202 includes a plurality of valves, including the IC circulation valve 226 , the reagent valve 228 , the IC media valve 230 , the air removal valve 232 , the cell inlet valve 234 , the wash valve 236 , the distribution valve 238 , the EC media valve 240 , the IC waste valve 242 , the EC waste valve 244 , and the harvest valve 246 .
  • Several sensors are also associated with the back portion 206 of the cell expansion machine 202 , including the IC outlet pressure sensor 248 , the combination IC inlet pressure and temperature sensors 250 , the combination EC inlet pressure and temperature sensors 252 , and the EC outlet pressure sensor 254 . Also shown is an optical sensor 256 for an air removal chamber.
  • a shaft or rocker control 258 for rotating the bioreactor 100 is shown.
  • Shaft fitting 260 associated with the shaft or rocker control 258 allows for proper alignment of a shaft access aperture, see e.g., 424 ( FIG. 4 ) of a tubing-organizer, see e.g., 300 ( FIG. 4 ) of a premounted conveyance assembly 210 or 400 with the back portion 206 of the cell expansion machine 202 .
  • Rotation of shaft or rocker control 258 imparts rotational movement to shaft fitting 260 and bioreactor 100 .
  • the alignment is a relatively simple matter of properly orienting the shaft access aperture 424 ( FIG. 4 ) of the premounted fluid conveyance assembly 210 or 400 with the shaft fitting 260 .
  • FIG. 4 a perspective view of a detachably-attachable premounted fluid conveyance assembly 400 is shown.
  • the premounted fluid conveyance assembly 400 may be detachably-attachable to the cell expansion machine 202 to facilitate relatively quick exchange of a new or unused premounted fluid conveyance assembly 400 at a cell expansion machine 202 for a used premounted fluid conveyance assembly 400 at the same cell expansion machine 202 .
  • the bioreactor 100 may be attached to a bioreactor coupling that includes a shaft fitting 402 .
  • the shaft fitting 402 includes one or more shaft fastening mechanisms, such as a biased arm or spring member 404 for engaging a shaft, e.g., 258 (shown in FIG. 3 ), of the cell expansion machine 202 .
  • the premounted fluid conveyance assembly 400 includes tubing 408 A, 408 B, 408 C, 408 D, 408 E, etc., and various tubing fittings to provide the fluid paths shown in FIGS. 5-9 , as discussed below. Pump loops 406 A and 406 B are also provided for the pump(s).
  • the various media may be provided at the site where the cell expansion machine 202 is located, the premounted fluid conveyance assembly 400 may include sufficient tubing length to extend to the exterior of the cell expansion machine 202 and to enable welded connections to tubing associated with the media bags, according to embodiments.
  • FIG. 5 illustrates a schematic of an embodiment of a cell expansion system 500
  • FIG. 6 illustrates a schematic of another embodiment of a cell expansion system 600 .
  • the cells are grown in the IC space.
  • the disclosure is not limited to such examples and may in other embodiments provide for cells to be grown in the EC space.
  • FIG. 5 illustrates a CES 500 , which includes first fluid circulation path 502 (also referred to as the “intracapillary loop” or “IC loop”) and second fluid circulation path 504 (also referred to as the “extracapillary loop” or “EC loop”), according to embodiments.
  • First fluid flow path 506 may be fluidly associated with cell growth chamber 501 to form first fluid circulation path 502 . Fluid flows into cell growth chamber 501 through IC inlet port 501 A, through hollow fibers in cell growth chamber 501 , and exits via IC outlet port 501 B. Pressure gauge 510 measures the pressure of media leaving cell growth chamber 501 . Media flows through IC circulation pump 512 which may be used to control the rate of media flow.
  • IC circulation pump 512 may pump the fluid in a first direction or second direction opposite the first direction. Exit port 501 B may be used as an inlet in the reverse direction. Media entering the IC loop 502 may enter through valve 514 . As those skilled in the art will appreciate, additional valves and/or other devices may be placed at various locations to isolate and/or measure characteristics of the media along portions of the fluid paths. Accordingly, it is to be understood that the schematic shown represents one possible configuration for various elements of the CES 500 , and modifications to the schematic shown are within the scope of the one or more present embodiments.
  • samples of media may be obtained from sample port 516 or sample coil 518 during operation.
  • Pressure/temperature gauge 520 disposed in first fluid circulation path 502 allows detection of media pressure and temperature during operation.
  • Media then returns to IC inlet port 501 A to complete fluid circulation path 502 .
  • Cells grown/expanded in cell growth chamber 501 may be flushed out of cell growth chamber 501 into harvest bag 599 through valve 598 or redistributed within the hollow fibers for further growth. This will be described in more detail below.
  • Fluid in second fluid circulation path 504 enters cell growth chamber 501 via EC inlet port 501 C, and leaves cell growth chamber 501 via EC outlet port 501 D.
  • Media in the EC loop 504 may be in contact with the outside of the hollow fibers in the cell growth chamber 501 , thereby allowing diffusion of small molecules into and out of the hollow fibers.
  • Pressure/temperature gauge 524 disposed in the second fluid circulation path 504 allows the pressure and temperature of media to be measured before the media enters the EC space of the cell growth chamber 501 .
  • Pressure gauge 526 allows the pressure of media in the second fluid circulation path 504 to be measured after it leaves the cell growth chamber 501 .
  • samples of media may be obtained from sample port 530 or a sample coil during operation.
  • fluid in second fluid circulation path 504 passes through EC circulation pump 528 to oxygenator or gas transfer module 532 .
  • EC circulation pump 528 may also pump the fluid in opposing directions.
  • Second fluid flow path 522 may be fluidly associated with oxygenator or gas transfer module 532 via oxygenator inlet port 534 and oxygenator outlet port 536 .
  • fluid media flows into oxygenator or gas transfer module 532 via oxygenator inlet port 534 , and exits oxygenator or gas transfer module 532 via oxygenator outlet port 536 .
  • Oxygenator or gas transfer module 532 adds oxygen to and removes bubbles from media in the CES 500 .
  • media in second fluid circulation path 504 may be in equilibrium with gas entering oxygenator or gas transfer module 532 .
  • the oxygenator or gas transfer module 532 may be any appropriately sized oxygenator or gas transfer device. Air or gas flows into oxygenator or gas transfer module 532 via filter 538 and out of oxygenator or gas transfer device 532 through filter 540 . Filters 538 and 540 reduce or prevent contamination of oxygenator or gas transfer module 532 and associated media. Air or gas purged from the CES 500 during portions of a priming sequence may vent to the atmosphere via the oxygenator or gas transfer module 532 .
  • first fluid circulation path 502 and second fluid circulation path 504 flows through cell growth chamber 501 in the same direction (a co-current configuration).
  • the CES 500 may also be configured to flow in a counter-current conformation.
  • media including cells (from bag 562 ), and fluid media from bag 546 may be introduced to first fluid circulation path 502 via first fluid flow path 506 .
  • Fluid container 562 e.g., Cell Inlet Bag or Saline Priming Fluid for priming air out of the system
  • valve 564 may be fluidly associated with the first fluid flow path 506 and the first fluid circulation path 502 via valve 564 .
  • Fluid containers, or media bags, 544 e.g., Reagent
  • 546 e.g., IC Media
  • First and second sterile sealable input priming paths 508 and 509 are also provided.
  • An air removal chamber (ARC) 556 may be fluidly associated with first circulation path 502 .
  • the air removal chamber 556 may include one or more ultrasonic sensors including an upper sensor and lower sensor to detect air, a lack of fluid, and/or a gas/fluid interface, e.g., an air/fluid interface, at certain measuring positions within the air removal chamber 556 .
  • ultrasonic sensors may be used near the bottom and/or near the top of the air removal chamber 556 to detect air, fluid, and/or an air/fluid interface at these locations.
  • Embodiments provide for the use of numerous other types of sensors without departing from the spirit and scope of the present disclosure.
  • optical sensors may be used in accordance with embodiments of the present disclosure. Air or gas purged from the CES 500 during portions of the priming sequence or other protocols may vent to the atmosphere out air valve 560 via line 558 that may be fluidly associated with air removal chamber 556 .
  • Fluid container 566 may be fluidly associated with valve 570 that may be fluidly associated with first fluid circulation path 502 via distribution valve 572 and first fluid inlet path 542 .
  • fluid container 566 may be fluidly associated with second fluid circulation path 504 via second fluid inlet path 574 and EC inlet path 584 by opening valve 570 and closing distribution valve 572 .
  • fluid container 568 may be fluidly associated with valve 576 that may be fluidly associated with first fluid circulation path 502 via first fluid inlet path 542 and distribution valve 572 .
  • fluid container 568 may be fluidly associated with second fluid inlet path 574 by opening valve 576 and closing valve distribution 572 .
  • An optional heat exchanger 552 may be provided for media reagent or wash solution introduction.
  • fluid may be initially advanced by the IC inlet pump 554 .
  • fluid may be initially advanced by the EC inlet pump 578 .
  • An air detector 580 such as an ultrasonic sensor, may also be associated with the EC inlet path 584 .
  • first and second fluid circulation paths 502 and 504 are connected to waste line 588 .
  • IC media may flow through waste line 588 and to waste or outlet bag 586 .
  • EC media may flow through waste line 588 to waste or outlet bag 586 .
  • cells may be harvested via cell harvest path 596 .
  • cells from cell growth chamber 501 may be harvested by pumping the IC media containing the cells through cell harvest path 596 and valve 598 to cell harvest bag 599 .
  • Various components of the CES 500 may be contained or housed within a machine or housing, such as cell expansion machine 202 ( FIGS. 2 and 3 ), wherein the machine maintains cells and media at a predetermined temperature.
  • a machine or housing such as cell expansion machine 202 ( FIGS. 2 and 3 ), wherein the machine maintains cells and media at a predetermined temperature.
  • CES 600 includes a first fluid circulation path 602 (also referred to as the “intracapillary loop” or “IC loop”) and second fluid circulation path 604 (also referred to as the “extracapillary loop” or “EC loop”).
  • First fluid flow path 606 may be fluidly associated with cell growth chamber 601 to form first fluid circulation path 602 .
  • Fluid flows into cell growth chamber 601 through IC inlet port 601 A, through hollow fibers in cell growth chamber 601 , and exits via IC outlet port 601 B.
  • Pressure sensor 610 measures the pressure of media leaving cell growth chamber 601 .
  • sensor 610 may, in embodiments, also be a temperature sensor that detects the media pressure and temperature during operation.
  • IC circulation pump 612 may pump the fluid in a first direction or second direction opposite the first direction.
  • Exit port 601 B may be used as an inlet in the reverse direction.
  • Media entering the IC loop may enter through valve 614 .
  • additional valves and/or other devices may be placed at various locations to isolate and/or measure characteristics of the media along portions of the fluid paths. Accordingly, it is to be understood that the schematic shown represents one possible configuration for various elements of the CES 600 , and modifications to the schematic shown are within the scope of the one or more present embodiments.
  • samples of media may be obtained from sample coil 618 during operation. Media then returns to IC inlet port 601 A to complete fluid circulation path 602 . Cells grown/expanded in cell growth chamber 601 may be flushed out of cell growth chamber 601 into harvest bag 699 through valve 698 and line 697 . Alternatively, when valve 698 is closed, the cells may be redistributed within chamber 601 for further growth.
  • Fluid in second fluid circulation path 604 enters cell growth chamber 601 via EC inlet port 601 C and leaves cell growth chamber 601 via EC outlet port 601 D.
  • Media in the EC loop may be in contact with the outside of the hollow fibers in the cell growth chamber 601 , thereby allowing diffusion of small molecules into and out of the hollow fibers that may be within chamber 601 , according to an embodiment.
  • Pressure/temperature sensor 624 disposed in the second fluid circulation path 604 allows the pressure and temperature of media to be measured before the media enters the EC space of the cell growth chamber 601 .
  • Sensor 626 allows the pressure and/or temperature of media in the second fluid circulation path 604 to be measured after it leaves the cell growth chamber 601 .
  • samples of media may be obtained from sample port 630 or a sample coil during operation.
  • Second fluid flow path 622 may be fluidly associated with oxygenator or gas transfer module 632 via an inlet port 632 A and an outlet port 632 B of oxygenator or gas transfer module 632 .
  • fluid media flows into oxygenator or gas transfer module 632 via inlet port 632 A, and exits oxygenator or gas transfer module 632 via outlet port 632 B.
  • Oxygenator or gas transfer module 632 adds oxygen to and removes bubbles from media in the CES 600 .
  • media in second fluid circulation path 604 may be in equilibrium with gas entering oxygenator or gas transfer module 632 .
  • the oxygenator or gas transfer module 632 may be any appropriately sized device useful for oxygenation or gas transfer. Air or gas flows into oxygenator or gas transfer module 632 via filter 638 and out of oxygenator or gas transfer device 632 through filter 640 . Filters 638 and 640 reduce or prevent contamination of oxygenator or gas transfer module 632 and associated media. Air or gas purged from the CES 600 during portions of a priming sequence may vent to the atmosphere via the oxygenator or gas transfer module 632 .
  • first fluid circulation path 602 and second fluid circulation path 604 flows through cell growth chamber 601 in the same direction (a co-current configuration).
  • the CES 600 may also be configured to flow in a counter-current conformation, according to embodiments.
  • media including cells (from a source such as a cell container, e.g. a bag) may be attached at attachment point 662 , and fluid media from a media source may be attached at attachment point 646 .
  • the cells and media may be introduced into first fluid circulation path 602 via first fluid flow path 606 .
  • Attachment point 662 may be fluidly associated with the first fluid flow path 606 via valve 664
  • attachment point 646 may be fluidly associated with the first fluid flow path 606 via valve 650 .
  • a reagent source may be fluidly connected to point 644 and be associated with fluid inlet path 642 via valve 648 , or second fluid inlet path 674 via valves 648 and 672 .
  • Air removal chamber (ARC) 656 may be fluidly associated with first circulation path 602 .
  • the air removal chamber 656 may include one or more sensors including an upper sensor and lower sensor to detect air, a lack of fluid, and/or a gas/fluid interface, e.g., an air/fluid interface, at certain measuring positions within the air removal chamber 656 .
  • ultrasonic sensors may be used near the bottom and/or near the top of the air removal chamber 656 to detect air, fluid, and/or an air/fluid interface at these locations.
  • Embodiments provide for the use of numerous other types of sensors without departing from the spirit and scope of the present disclosure.
  • optical sensors may be used in accordance with embodiments of the present disclosure. Air or gas purged from the CES 600 during portions of a priming sequence or other protocol(s) may vent to the atmosphere out air valve 660 via line 658 that may be fluidly associated with air removal chamber 656 .
  • Attachment point 666 may be fluidly associated with valve 670 that may be fluidly associated with first fluid circulation path 602 via valve 672 and first fluid inlet path 642 .
  • attachment point 666 may be fluidly associated with second fluid circulation path 604 via second fluid inlet path 674 and second fluid flow path 684 by opening valve 670 and closing valve 672 .
  • attachment point 668 may be fluidly associated with valve 676 that may be fluidly associated with first fluid circulation path 602 via first fluid inlet path 642 and valve 672 .
  • fluid container 668 may be fluidly associated with second fluid inlet path 674 by opening valve 676 and closing valve distribution 672 .
  • fluid may be initially advanced by the IC inlet pump 654 .
  • fluid may be initially advanced by the EC inlet pump 678 .
  • An air detector 680 such as an ultrasonic sensor, may also be associated with the EC inlet path 684 .
  • first and second fluid circulation paths 602 and 604 are connected to waste line 688 .
  • IC media may flow through waste line 688 and to waste or outlet bag 686 .
  • EC media may flow to waste or outlet bag 686 .
  • cells from cell growth chamber 601 may be harvested via cell harvest path 697 .
  • cells from cell growth chamber 601 may be harvested by pumping the IC media containing the cells through cell harvest path 697 , with valve 698 open, into cell harvest bag 699 .
  • CES 600 may be contained or housed within a machine or housing, such as cell expansion machine 202 ( FIGS. 2 and 3 ), wherein the machine maintains cells and media at a predetermined temperature. It is further noted that, in embodiments, components of CES 600 and CES 500 ( FIG. 5 ) may be combined. In other embodiments, a CES may include fewer or additional components than those shown in FIGS. 5 and 6 and still be within the scope of the present disclosure.
  • FIGS. 5 and 6 illustrate schematics of different embodiments of cell expansion systems
  • FIGS. 7 and 8 depict these same cell expansion systems with the waste or outlet bags ( 586 and 686 ) replaced by media bags in accordance with embodiments of the present disclosure.
  • waste or outlet bag 686 in CES 600 FIG. 6
  • media e.g., base media, bag 700
  • one or more pumps e.g., IC Circulation Pump 612 , EC Inlet Pump 678 , and IC Inlet Pump 654 , have been turned “OFF,” according to an embodiment. There is thus no active inlet fluid flow into cell growth chamber 601 .
  • the EC Circulation Pump 628 is left “ON” and the EC Waste Valve 692 is left “OPEN.”
  • This configuration allows fluid from media bag 700 to backflow into the CES 600 system at a rate equal to the rate of evaporation from the oxygenator or gas transfer module 632 .
  • the fluid lost in the system due to evaporation may thus be replaced without diluting chemical signaling occurring in the bioreactor 601 during cell growth therein.
  • the lag phase of cell growth in the bioreactor 601 may therefore be significantly reduced, and more efficient cell expansion may occur.
  • FIG. 7 shows an embodiment in which the IC Circulation Pump 612 , EC Inlet Pump 678 , and IC Inlet Pump 654 have been turned “OFF,” other embodiments provide for one or more of such pumps, e.g., the IC Circulation Pump 612 , for example, to remain “ON” or activated (not shown in FIG. 7 ). For example, it may be desired in embodiments to continue circulation in the intracapillary side depending on the type of cells, e.g., non-adherent cells, being expanded, according to an embodiment.
  • the media bag (e.g., 700 ) may be positioned at a physically higher level than at least a portion of the EC loop 604 to allow gravity to assist in draining fluid from the media bag into the EC loop 604 .
  • the waste bag 686 ( FIG. 6 ) may be positioned lower than the EC loop 604 to allow gravity to assist in draining waste media into the waste bag 686 .
  • the substitute or replacement media bag 700 may be positioned physically higher than the original position of the waste bag 686 .
  • FIG. 8 a similar configuration is shown, in which, for example, waste bag 586 has been replaced by media, e.g., base media, bag 800 . Further, one or more pumps, e.g., IC Circulation Pump 512 , EC Inlet Pump 578 , and IC Inlet Pump 554 , have been turned “OFF,” according to an embodiment. There is thus no active inlet fluid flow into the bioreactor 501 .
  • the EC Circulation Pump 528 may be left “ON,” and the EC Waste Valve 582 may be left “OPEN.”
  • such configuration allows fluid from the substitute or replacement media bag 800 to backflow into the system at a rate equal to the rate of evaporation from the gas transfer module or oxygenator 532 .
  • the fluid lost in the system due to evaporation may thus be replaced without diluting chemical signaling occurring in the bioreactor 501 during cell growth therein.
  • the lag phase of cell growth in the bioreactor 501 may therefore be significantly reduced, and more efficient cell expansion may occur.
  • FIG. 8 shows an embodiment in which the IC Circulation Pump 512 , EC Inlet Pump 578 , and IC Inlet Pump 554 have been turned “OFF,” other embodiments provide for one or more of such pumps, such as the IC Circulation Pump 512 , for example, to remain “ON” or activated (not shown in FIG. 8 ). For example, it may be desired in embodiments to continue circulation in the intracapillary side depending on the type of cells, e.g., non-adherent cells, being expanded, according to an embodiment.
  • the substitute or replacement media bag may be positioned physically higher than the original position of the waste bag 586 to allow gravity to assist in draining media into the EC loop 504 .
  • the replacement of the waste bag with a media bag allows passive replacement of fluid lost due to evaporation.
  • passive replacement of fluid may provide a significant conservation of fluid in cell expansion processes.
  • media may be added and circulated in the IC loop during attachment of cells to replace fluid lost due to evaporation.
  • an excess amount over the amount that has evaporated
  • the media may include expensive additives. Saving about 130 mL/day, for example, may provide significant cost savings over other cell expansion processes.
  • FIG. 9 illustrates an embodiment in which a molecule source, e.g., a cell signaling protein molecule source, may be added to a cell expansion system, such as CES 600 ( FIG. 6 ) (or CES 500 ( FIG. 5 )), for example.
  • the molecule source 900 may be a cytokine source welded into the sample coil or sampling coil 618 , in which such cytokine source comprises a piece of tubing or other material welded into the sampling coil 618 .
  • cytokines may be added to the IC loop 602 without diluting such proteins, in which such dilution may occur where the cytokines are added instead at an IC Media bag, for example.
  • the molecules are directly added to the IC loop 602 .
  • Such direct addition may also occur at a sample port, for example, according to an embodiment.
  • Cytokines in the cell growth chamber 601 may thus be passively or actively replenished by such cytokine source.
  • the IC Circulation Pump 612 is turned to the “ON” position to allow the cytokines entering the IC loop 602 at the sampling coil 618 to be pumped to the expanding cell population in the bioreactor 601 .
  • Such cell source may ultimately save significant resources where chemical-signaling proteins used in the bioreactor are particularly costly, e.g., cytokines.
  • FIG. 10 illustrates example operational steps 1000 for passively replacing fluid to control chemical signaling in a closed cell expansion system, in accordance with embodiments of the present disclosure.
  • START operation 1002 is initiated, and process 1000 proceeds to load the disposable tubing set 1004 onto the cell expansion system.
  • the system is primed 1006 , such as by having a user or operator instruct the system to prime by selecting a task for priming, for example.
  • the system is primed 1006 automatically without any selection of a task or instruction from a user or operator.
  • process 1000 proceeds to coat the bioreactor 1008 , in which the bioreactor is coated with a reagent.
  • a reagent is loaded into the IC loop until the Reagent Bag is empty.
  • the reagent is chased from the air removal chamber into the IC loop, and the reagent is then circulated in the IC loop.
  • the IC/EC Washout task is executed 1010 , in which fluid on the IC circulation loop and on the EC circulation loop is replaced.
  • the replacement volume is determined by the number of IC Volumes and EC Volumes exchanged, according to an embodiment.
  • the condition media task 1012 is executed to allow the media to reach equilibrium with the provided gas supply before cells are loaded into the bioreactor.
  • the system is then maintained in a proper state until a user or operator is ready to load cells into the bioreactor.
  • a user or operator may not be needed to perform the noted steps/operations; rather, the steps/operations may be performed automatically by the cell expansion system.
  • Process 1000 next proceeds to loading cells into the bioreactor from a cell inlet bag with circulating distribution 1014 .
  • cells are loaded into the bioreactor from the cell inlet bag until the bag is empty.
  • Cells are then chased from the air removal chamber to the bioreactor. Larger chase volumes spread the cells and move the cells toward the IC outlet.
  • the distribution of cells is promoted across the membrane via IC circulation, such as through the IC circulation pump, with no IC inlet, for example.
  • the waste or outlet bag is replaced with a media bag 1016 .
  • the media bag comprises about 500 mL of base media.
  • the media bag may comprise other fluids and/or constituents, according to embodiments.
  • the replacement of the outlet or waste bag with a media bag 1016 may be optional, in which fluid may be passively replaced by interrupting protocol procedures being executed and allowing any fluid in the outlet or waste bag (assuming no constituents toxic to cell growth are present in the outlet or waste bag) to be passively added to the system at the rate of evaporation during conditions of no active inlet fluid flow. Such passive addition of fluid avoids adding an excess amount of fluid and, thus, avoids diluting chemical signaling molecules.
  • one or more pumps may then be turned “OFF” or may otherwise be indicated to stop or deactivate 1018 .
  • Any adherent cells in the bioreactor are then allowed to attach to the bioreactor membrane 1020 for a period of time, such as for about eighteen (18) to about twenty-four (24) hours, according to an embodiment of the present disclosure.
  • flow continues on the EC circulation loop, in which the EC circulation rate is maintained at about 30 mL/min, according to an embodiment.
  • a non-zero EC circulation rate helps to maintain the proper or desired gas concentration across the fibers of the bioreactor membrane by continuing to pump fluid in the EC loop through the gas transfer module or oxygenator. While the proper or desired gas concentration is maintained through the use of the gas transfer module, evaporation of fluid also occurs at the gas transfer module.
  • media from the media bag may back-flow into the system and be pumped by the EC Circulation Pump through the EC loop. The media may thus replace fluid lost due to evaporation from the gas transfer module at the rate of evaporation.
  • membrane fibers in the bioreactor will not be diluted with excess fluid, and the transition of cell growth out of the lag phase will not be inhibited.
  • a continued cell attachment phase 1022 continues for up to about forty-eight (48) hours.
  • the IC circulation pump may be activated or turned “ON” to provide even the furthest fibers of the bioreactor membrane with media.
  • the IC circulation pump may be activated to adjust the IC circulation rate to about 20 mL/min, according to an embodiment of the present disclosure.
  • the IC inlet rate remains at 0 mL/min.
  • the substitute media bag in replacement of the waste bag continues to provide any necessary fluid replacement to the system while not diluting the membranes or inhibiting chemical signaling.
  • Operation 1022 with modified feeding of the cells thus allows for cell attachment to continue without disruption of chemical signaling occurring in the bioreactor.
  • This continued cell attachment phase continues, according to embodiments, for up to about forty-eight (48) additional hours and/or, in embodiments, until the lactate generation of the cells is greater than or equal to about 6 mmol/L.
  • the concentration of lactate is measured.
  • the lactate generation rate for example, is measured.
  • the lactate generation is thus checked at operation 1024 to determine if the concentration of lactate is equal to or exceeds 6 mmol/L.
  • the lactate generation is checked at operation 1024 to determine the concentration of lactate in relation to another predetermined amount.
  • Process 1000 next proceeds to query 1026 , in which it is determined whether more than forty-eight hours has passed since the IC circulation pump was activated or whether the concentration of lactate is equal to or greater than about 6 mmol/L. If less than forty-eight (48) hours has passed or if the concentration of lactate is not equal to or in excess of about 6 mmol/L, process 1000 proceeds NO to check lactate generation operation 1024 and then to query 1026 again. It is noted that the present disclosure is not limited to determining whether forty-eight (48) hours have passed or whether there is a lactate concentration equal to or in excess of 6 mmol/L. In other embodiments, process 1000 may involve a different predetermined period of time.
  • a determination may be made whether about 12 hours, about 24 hours, about 36 hours, or about 40 hours have passed.
  • the predetermined period of time may be about 50 hours or about 60 hours.
  • a determination may be made whether more than about 12 hours, more than about 24 hours, more than about 36 hours, or more than about 40 hours have passed.
  • a determination may be made whether less than about 60 hours or less than about 50 hours have passed.
  • process 1000 may involve determining whether the concentration of lactate is equal to or greater than another predetermined amount, such as about 3 mmol/L, about 4 mmol/L, about 5 mmol/L, about 7 mmol/L, or about 8 mmol/L.
  • a determination may be made whether the concentration of lactate is more than about 3 mmol/L, more than about 4 mmol/L, or more than about 5 mmol/L. In other embodiments, a determination may be made whether the concentration of lactate is less than about 8 mmol/L or less than about 7 mmol/L.
  • process 1000 proceeds YES to feed cells operation 1028 , in which the IC inlet pump is activated or turned “ON” to maintain an IC Inlet Rate of 0.1 mL/min.
  • process 1000 proceeds to measure the glucose consumption 1030 .
  • the concentration of glucose is measured.
  • the glucose consumption rate for example, is measured.
  • it is determined whether the measured glucose consumption is less than about 70 mg/L, in an embodiment.
  • process 1000 proceeds YES to double the IC Inlet Rate 1034 .
  • Process 1000 then proceeds to operation 1030 to continue measuring the glucose consumption of the cells and back to query 1032 .
  • process 1000 may involve a different predetermined amount. For example, in embodiments, process 1000 may involve determining whether the glucose consumption is less than another predetermined amount, such as about 65 mg/L, about 60 mg/L, or about 55 mg/L, for example. In other embodiments, the process 1000 may involve determining whether the glucose consumption is less than another predetermined amount, such as about 85 mg/L, about 80 mg/L, or about 75 mg/L, for example. In embodiments, a determination may be made whether the glucose consumption is more than about 55 mg/L, more than about 60 mg/L, or more than about 65 mg/L. In other embodiments, a determination may be made whether the glucose consumption is less than about 85 mg/L, less than about 80 mg/L, or less than about 75 mg/L.
  • process 1000 proceeds NO to release the cells operation 1036 , in which the cells are released from the membrane of the bioreactor and are suspended in the IC loop.
  • an IC/EC Washout task in preparation for adding a reagent is performed.
  • IC/EC media may be replaced with a phosphate buffered saline (PBS) to remove protein, calcium (Ca 2+ ), and magnesium (Mg 2+ ) in preparation for adding trypsin, or another chemical-releasing agent, to release any adherent cells.
  • PBS phosphate buffered saline
  • Mg 2+ magnesium
  • a reagent may be loaded into the system until the reagent bag is empty.
  • the reagent may be chased into the IC loop, and the reagent may be mixed within the IC loop.
  • harvest operation 1038 transfers the cells in suspension from the IC circulation loop, including any cells remaining in the bioreactor, to the harvest bag.
  • Process 1000 then terminates at END operation 1040 .
  • FIG. 11 depicts a flow diagram illustrating the operational characteristics of a process 1100 for adding a molecule from a molecule source, implemented as part of a cell expansion system itself, in accordance with embodiments of the present disclosure. While various example embodiments of a cell expansion system and methods for adding a molecule to a cell expansion system have been described, FIG. 11 illustrates example operational steps 1100 for adding a molecule that affects chemical signaling in a closed cell expansion system, in accordance with embodiments of the present disclosure. Some embodiments provide for the passive addition of a molecule from a molecule source. START operation 1102 is initiated, and process 1100 proceeds to load a disposable tubing set 1104 onto the cell expansion system.
  • the system is primed 1106 , such as by having an operator or user provide an instruction to the system to prime by selecting a task for priming, for example.
  • the system is primed 1106 automatically without any selection of a task or instruction from an operator or user.
  • process 1100 proceeds to coat the bioreactor 1108 , in which the bioreactor may be coated with a reagent.
  • a reagent is loaded into the IC loop until a reagent container is empty.
  • the reagent may be chased from the air removal chamber into the IC loop, and the reagent may then be circulated in the IC loop.
  • the IC/EC Washout task may be executed 1110 , in which fluid on the IC circulation loop and on the EC circulation loop may be replaced, according to an embodiment.
  • the replacement volume is determined by the number of IC Volumes and EC Volumes exchanged.
  • condition media task 1112 is executed to allow the media to reach equilibrium with the provided gas supply before cells are loaded into the bioreactor. For example, rapid contact between the media and the gas supply provided by the gas transfer module or oxygenator is provided by using a high EC circulation rate.
  • the system may then be maintained in a proper or desired state until an operator or user is ready to load cells into the bioreactor. In embodiments, such loading of cells is performed automatically.
  • Process 1100 next proceeds to loading cells into the bioreactor from a cell inlet bag with circulating distribution 1114 .
  • cells are loaded into the bioreactor from a cell inlet bag until the bag is empty.
  • Cells are then chased from the air removal chamber to the bioreactor.
  • cells are spread and move toward the IC outlet.
  • the distribution of cells may be promoted across the membrane via IC circulation, such as through the IC circulation pump, with no IC inlet flow, for example.
  • the waste bag is replaced with a media bag 1116 .
  • the media bag comprises about 500 mL of base media.
  • the media bag comprises any type of replacement fluid.
  • step 1116 is optional, in which the outlet or waste bag stays connected and is not replaced with another bag.
  • step 1116 is optional, in which the outlet or waste bag stays connected and desired constituents or other fluid(s) are added to the outlet or waste bag for passively adding such constituents/other fluid to the system.
  • one or more pumps may then be turned “OFF” or may otherwise be indicated to stop or deactivate 1118 .
  • Any adherent cells in the bioreactor are then allowed to attach to the bioreactor membrane 1120 for a period of time, such as for about eighteen (18) to about twenty-four (24) hours, according to an embodiment of the present disclosure.
  • flow may continue on the EC circulation loop, in which the EC circulation rate may be maintained at about 30 mL/min, according to an embodiment.
  • a non-zero EC circulation rate helps to maintain the proper or desired gas concentration across the fibers of the bioreactor membrane by continuing to pump fluid in the EC loop through the gas transfer module or oxygenator. While the proper or desired gas concentration is maintained through the use of the gas transfer module, evaporation of fluid also occurs at the gas transfer module.
  • media from the substitute media bag may back-flow into the system and be pumped by the EC Circulation Pump through the EC loop. The media may thus replace fluid lost due to evaporation from the gas transfer module at the rate of evaporation.
  • membrane fibers in the bioreactor will not be diluted with excess fluid, and the transition of cell growth out of the lag phase will not be inhibited.
  • the molecule may be a protein molecule that is added to promote expansion of the cells.
  • the molecule may be a signaling molecule, such as one or more cytokines or growth factors that are involved in intercellular communications.
  • the molecule may signal the cells to expand.
  • the molecule may not be directly involved in signaling but may help create an environment that is conducive to cell growth, in which examples of such molecules include carrier proteins, buffers, pH modifiers, etc.
  • the molecule is added to the space where the cells are being grown, e.g., the IC or EC space.
  • the molecules are added directly to the IC loop from a direct source of such molecules.
  • Such direct addition may occur at a sampling coil or at a sample port, for example, according to embodiments.
  • Cytokines, or other type of cell-signaling protein molecules may be added to the bioreactor by, for example, welding a tubing line or other material connected to a cytokine source to a sampling coil or sample coil of the cell expansion system. The cytokines may thus be added to the bioreactor at the sample coil.
  • Such direct addition results in a significant savings of cytokines, which may be costly, because a much higher amount of cytokines would need to be added to a media bag to compensate for dilution of the cytokines by the media than are needed when only the cytokine source itself replenishes the bioreactor, according to an embodiment.
  • cytokines tend to degrade quickly, in which such degradation may be minimized by adding cytokines closer to the expanding cell population, e.g., at the sample coil of the bioreactor itself.
  • the cytokines in the bioreactor may thus be maintained at a certain level while conserving resources.
  • a source i.e., direct source
  • cytokines may be added to the IC loop without diluting such proteins, in which such dilution may occur where the cytokines are added instead at the IC Media bag, for example.
  • the add molecule phase 1122 may be performed after the waste bag is replaced with a media bag 1116 , according to an embodiment.
  • the molecule that is added at operation 1122 may be relatively expensive, and it is desirable to use the minimum amount required to promote growth of the cells.
  • Performing operation 1116 first allows media from the media bag (replacing the waste bag) to back-flow into the system and be pumped by the EC Circulation Pump through the EC loop.
  • only the media that is lost due to evaporation from the gas transfer module is replaced and at the rate of evaporation.
  • the molecule may not be diluted with excess fluid. Accordingly, in an embodiment, only an amount of the molecule that may be effective at promoting growth may be added at operation 1122 since dilution by excess fluid may not be occurring.
  • operation 1124 may involve a number of sub-operations.
  • the sub-operations include operations performed in process 1000 ( FIG. 10 ).
  • a circulating media operation may be performed to feed the cells.
  • the IC circulation pump may be activated or turned “ON” to provide even the furthest fibers of the bioreactor membrane with media.
  • the IC circulation pump may be activated to adjust the IC circulation rate to about 20 mL/min, according to an embodiment of the present disclosure. In some embodiments, even though the IC circulation pump is turned on, the IC inlet rate remains at 0 mL/min.
  • operation 1124 allows cell attachment and cell growth to occur without disruption of chemical signaling by dilution of the molecules. This continued cell attachment and growth may continue, according to embodiments, for some predetermined period of time or may be based on a lactate generation of the cells, e.g., 6 mmol/L (in an embodiment). In these embodiments, additional sub-operations, such as determining lactate concentration(s) or that a predetermined period of time has elapsed, may be performed.
  • Operation 1124 may further involve a sub-operation of activating the IC inlet pump to maintain a predetermined IC inlet rate, e.g., 0.1 mL/min. This sub-operation may be triggered based on a predetermined period of time having elapsed or on a measurement, such as lactate concentration, for example.
  • operation 1124 may involve a number of sub-operations to determine when to stop growing cells and begin releasing and harvesting cells. In one embodiment, this may include measuring a parameter, such as glucose consumption. In some embodiments, a predetermined glucose concentration, e.g., greater than 70 mg/L, may trigger subsequent operations, e.g., 1126 and 1128 . In other embodiments, other parameters or the passage of a predetermined period of time may trigger subsequent operations.
  • a parameter such as glucose consumption.
  • a predetermined glucose concentration e.g., greater than 70 mg/L
  • subsequent operations e.g., 1126 and 1128 .
  • other parameters or the passage of a predetermined period of time may trigger subsequent operations.
  • any adherent cells are released from the membrane of the bioreactor and are suspended, e.g., in the IC loop.
  • an IC/EC washout task in preparation for adding a reagent to release the cells may be performed as part of operation 1126 .
  • IC/EC media may be replaced with PBS to remove protein, calcium (Ca 2+ ), and magnesium (Mg 2+ ) in preparation for adding trypsin, or other chemical-releasing agent, to release any adherent cells.
  • a reagent may be loaded into the system until the reagent bag is empty. The reagent may be chased into the IC loop, and the reagent may be mixed within the IC loop.
  • harvest operation 1128 transfers the cells in suspension from the IC circulation loop, including any cells remaining in the bioreactor, to a harvest bag(s).
  • Process 1100 then terminates at END operation 1130 .
  • START operation 1202 is initiated, and process 1200 proceeds to load the disposable tubing set 1204 onto a cell expansion system.
  • the system is primed 1206 , such as by having a user or operator instruct the system to prime by selecting a task for priming, for example.
  • the system is primed 1206 automatically without any selection of a task or instruction from a user or operator.
  • process 1200 proceeds to IC/EC washout 1208 , in which fluid on the IC and EC circulation loops may be replaced in preparation for cell culturing.
  • the replacement volume may be specified by the number of IC Volumes and EC Volumes exchanged, according to embodiments.
  • process 1200 proceeds to condition media task 1210 .
  • rapid contact between the media and the gas supply may be provided by using a high EC circulation rate.
  • the system may then be maintained in a proper state until the user or operator is ready to load cells into the bioreactor.
  • a user or operator may not be needed to perform the noted steps/operations; rather, the steps/operations may be performed automatically by the cell expansion system.
  • Process 1200 next proceeds to loading cells with uniform suspension 1212 .
  • cells may be loaded from a cell inlet bag.
  • IC circulation may be used to distribute the cells.
  • cells are loaded into the bioreactor from a cell inlet bag. Cells are then chased from the air removal chamber to the IC loop. The distribution of cells is promoted across the membrane via IC circulation with no IC inlet, for example, and thus no ultrafiltration, according to embodiments.
  • process 1200 proceeds to the optional (shown in a dashed-line format) step of replacing an outlet or waste bag with a media bag (e.g., a substitute media bag) 1214 .
  • a media bag e.g., a substitute media bag
  • the substitute media bag comprises about 0.2 L of media without protein.
  • Other volumes and types of replacement fluid in the substitute media bag may be used in accordance with embodiments of the present disclosure.
  • Process 1200 next proceeds to turning “OFF” or otherwise deactivating one or more pumps 1216 .
  • the IC inlet pump and the EC inlet pump are turned “OFF” or otherwise indicated to stop or deactivate 1216 .
  • Such pump deactivation allows chemical signals, such as CCK, to increase in concentration by turning the inlet media flow rate “OFF” to the IC circulation loop and the EC circulation loop.
  • fluid from the substitute bag may be passively added to the system at the rate of evaporation during conditions of no active inlet fluid flow.
  • fluid may be passively replaced in the system by interrupting protocol procedures being executed and allowing any fluid in the outlet or waste bag (assuming no constituents toxic to cell growth are present in the outlet or waste bag) to be passively added to the system at the rate of evaporation during conditions of no active inlet fluid flow.
  • Such passive addition of fluid avoids adding an excess amount of fluid and, thus, avoids diluting chemical signaling.
  • the EC circulation pump may remain “ON.” In further embodiments, both the IC circulation pump and the EC circulation pump remain activated or “ON.”
  • process 1200 proceeds to feeding the cells 1218 .
  • the cell culture may be sampled for cell counts as well by excising a length of tubing to provide a representative cell concentration sample of the IC loop.
  • cells may be counted by withdrawing a sample from the sampling coil or sample port, for example.
  • Process 1200 next proceeds to measuring the glucose and/or lactate concentration(s) 1220 .
  • process 1200 proceeds to feed the cells by adding a controlled flow rate to the IC circulation loop and/or the EC circulation loop 1226 once the cell culture conditions have reached a minimum tolerance glucose concentration or a maximum tolerance lactate concentration, for example.
  • a low flow rate is continuously added to the IC circulation loop and/or the EC circulation loop. Such feeding with the continuous addition of a low flow rate, for example, may occur earlier or later than day 4, according to embodiments.
  • Harvest operation 1228 next transfers cells in suspension from the IC circulation loop, including cells in the bioreactor, to a harvest bag.
  • Process 1200 then terminates at END operation 1230 .
  • an additional step(s) may include replacing the substitute media bag (previously used to replace the outlet or waste bag) with an outlet or waste bag.
  • Such outlet or waste bag may be the original outlet or waste bag used with the system, according to an embodiment.
  • process 1200 includes some optional steps/sub-steps shown with dashed-line format. However any steps listed above (in any of processes 1000 , 1100 , and/or 1200 ) that are not indicated as optional should not be considered as essential to the one or more present inventions, but may be performed in some embodiments of the one or more present inventions and not in others. Further, while some steps, operations and/or sub-operations are described with reference to an operator or user, such steps, operations and/or sub-operations may be performed automatically, according to embodiments.
  • FIG. 13 illustrates example components of a computing system 1300 upon which embodiments of the present disclosure may be implemented.
  • Computing system 1300 may be used in embodiments, for example, where a cell expansion system uses a processor to execute tasks, such as custom tasks or pre-programmed tasks performed as part of processes, such as processes 1000 , 1100 , and 1200 described above.
  • a pre-programmed task may include, “Feed Cells.”
  • the computing system 1300 may include a user interface 1302 , a processing system 1304 , and/or storage 1306 .
  • the user interface 1302 may include output device(s) 1308 , and/or input device(s) 1310 as understood by a person of skill in the art.
  • Output device(s) 1308 may include one or more touch screens, in which the touch screen may comprise a display area for providing one or more application windows.
  • the touch screen may also be an input device 1310 that may receive and/or capture physical touch events from a user or operator, for example.
  • the touch screen may comprise a liquid crystal display (LCD) having a capacitance structure that allows the processing system 1304 to deduce the location(s) of touch event(s), as understood by those of skill in the art.
  • LCD liquid crystal display
  • the processing system 1304 may then map the location of touch events to user interface (UI) elements rendered in predetermined locations of an application window.
  • UI user interface
  • the touch screen may also receive touch events through one or more other electronic structures, according to embodiments.
  • Other output devices 1308 may include a printer, speaker, etc.
  • Other input devices 1310 may include a keyboard, other touch input devices, mouse, voice input device, etc., as understood by a person of skill in the art.
  • Processing system 1304 may include a processing unit 1312 and/or a memory 1314 , according to embodiments of the present disclosure.
  • the processing unit 1312 may be a general purpose processor operable to execute instructions stored in memory 1314 .
  • Processing unit 1312 may include a single processor or multiple processors, according to embodiments. Further, in embodiments, each processor may be a multi-core processor having one or more cores to read and execute separate instructions.
  • the processors may include general purpose processors, application specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), other integrated circuits, etc., as understood by a person of skill in the art.
  • the memory 1314 may include any short-term or long-term storage for data and/or processor executable instructions, according to embodiments.
  • the memory 1314 may include, for example, Random Access Memory (RAM), Read-Only Memory (ROM), or Electrically Erasable Programmable Read-Only Memory (EEPROM), as understood by a person of skill in the art.
  • RAM Random Access Memory
  • ROM Read-Only Memory
  • EEPROM Electrically Erasable Programmable Read-Only Memory
  • Other storage media may include, for example, CD-ROM, tape, digital versatile disks (DVD) or other optical storage, tape, magnetic disk storage, magnetic tape, other magnetic storage devices, etc., as understood by a person of skill in the art.
  • Storage 1306 may be any long-term data storage device or component. Storage 1306 may include one or more of the systems described in conjunction with the memory 1314 , according to embodiments. The storage 1306 may be permanent or removable. In embodiments, storage 1306 stores data generated or provided by the processing system 1304 .
  • CES 500 FIG. 5
  • CES 600 FIG. 6
  • CES 700 FIG. 7
  • CES 800 FIG. 8
  • CES 900 FIG. 9
  • the example protocols including the steps (and any sub-steps) of loading cells and distributing cells, for example, may be performed automatically in some embodiments, such as by a processor executing pre-programmed tasks stored in memory.
  • the steps (and any sub-steps) are performed through the combination of automated and manual execution of operations.
  • the steps (and any sub-steps) are performed by an operator(s) or user(s) or through other manual means.
  • the bioreactor may include a hollow fiber membrane.
  • Table 1 describes the bags of solution that are attached to each line when performing the Coat Bioreactor portion of the protocol. These solutions and corresponding volumes are provided as one example of default settings that may be used.
  • This part of the example protocol is performed to replace the fluid on both the IC circulation loop and the EC circulation loop.
  • the replacement volume may be specified by the number of IC Volumes and EC Volumes exchanged.
  • Table 5 describes the bags of solution that are attached to each line when performing IC EC Washout of this example protocol. These solutions and corresponding volumes are provided as one example of default settings that may be used.
  • This part of the example protocol is performed to allow the media to reach equilibrium with the provided gas supply before loading the cells.
  • This task may include two separate steps:
  • Table 7 describes the bags of solution that are attached to each line when performing Condition Media. These solutions and corresponding volumes are provided as one example of default settings that may be used.
  • step 1 shown in Table 8 may be used.
  • step 2 shown in Table 9 may be used.
  • This part of the example protocol is performed to loads cells into the bioreactor from a cell inlet bag.
  • IC circulation may be used to distribute the cells and may not attempt to chase the cells from the line into the bioreactor. This task may include three separate steps.
  • Table 10 describes the bags of solution attached to each line when performing Load Cells with Circulating Distribution. These solutions and corresponding volumes are provided as one example of default settings that may be used.
  • step 1 shown in Table 11 may be used.
  • step 2 shown in Table 12 may be used.
  • step 3 shown in Table 13 may be used.
  • This part of the example protocol is performed to enable adherent cells to attach to the bioreactor membrane while allowing flow on the EC circulation loop.
  • the pump flow rate to the IC loop is set to approximately zero.
  • Table 14 describes the bags of solution attached to each line when performing Attach Cells. These solutions and corresponding volumes are provided as one example of default settings that may be used.
  • This part of the example protocol is performed to continuously add a low flow rate to the IC circulation loop and/or the EC circulation loop.
  • Table 16 describes the bags of solution attached to each line when performing Feed Cells. These solutions and corresponding volumes are provided as one example of default settings that may be used.
  • step 1 shown in Table 17 may be used.
  • the IC Inlet rate may be increased as needed. As one example, the IC inlet rate may be increased as follows: Day 1-Day 2: 0.0 mL/min; Day 2-Day 3: 0.1 mL/min; Day 3-Day 4: 0.2 mL/min; Day 4-Day 5: 0.4 mL/min; and Day 5-Day 6: 0.8 mL/min.
  • This part of the example protocol is performed to release cells from the membrane, leaving the cells in the IC loop.
  • Table 18 describes the bags of solution attached to each line when performing Release Adherent Cells. These solutions and corresponding volumes are provided as one example of default settings that may be used.
  • step 1 shown in Table 19 may be used.
  • step 2 shown in Table 20 may be used.
  • step 3 shown in Table 21 may be used.
  • step 4 shown in Table 22 may be used.
  • Samples may be taken from a sample coil and/or a sample port for a trypsin assay.
  • This part of the example protocol is performed to transfer cells in suspension from the IC circulation loop, including cells in the bioreactor, to the harvest bag.
  • Table 23 describes the bags of solution attached to each line when performing Harvest Cells. These solutions and corresponding volumes are provided as one example of default settings that may be used.
  • the values for Harvest Cells shown in Table 24 may be used.
  • Cholecystokinin is a regulatory hormone secreted by cells and, in many cases, may in part be responsible for cell culture maintenance and proliferation via chemical signaling. If CCK concentration in the culture media does not reach a threshold, the cell population can be compromised.
  • Example 2 provides an example of a cell-secreted chemical signal used to maintain and proliferate a population of cells in vitro; in this case, CHO cells.
  • the molecular weight of CCK of approximately 4,000 Daltons makes it small enough to readily pass through the microporous membrane of a hollow-fiber bioreactor.
  • dilution of the chemical signal may occur due to the freedom to pass through the membrane.
  • This part of the example protocol is performed to replace the fluid on both the IC circulation loop and the EC circulation loop in preparation for cell culturing.
  • the replacement volume may be specified by the number of IC Volumes and EC Volumes exchanged.
  • Table 25 describes the bags of solution that are attached to each line when performing IC EC Washout of this example protocol. These solutions and corresponding volumes are provided as one example of default settings that may be used.
  • This part of the example protocol is performed to allow the media to reach equilibrium with the provided gas supply before loading the cells.
  • This task may include two separate steps:
  • Table 27 describes the bags of solution that are attached to each line when performing Condition Media. These solutions and corresponding volumes are provided as one example of default settings that may be used.
  • step 1 shown in Table 28 may be used.
  • step 2 shown in Table 29 may be used.
  • This part of the example protocol is performed to load cells into the bioreactor from a cell inlet bag.
  • such cells comprise CHO cells.
  • IC circulation may be used to distribute the cells and may not attempt to chase the cells from the line into the bioreactor. This task may include three separate steps.
  • Table 30 describes the bags of solution attached to each line when performing Load Cells with Uniform Suspension. These solutions and corresponding volumes are provided as one example of default settings that may be used.
  • step 1 shown in Table 31 may be used.
  • step 2 shown in Table 32 may be used.
  • step 3 shown in Table 33 may be used.
  • This part of the example protocol is performed to allow chemical signals, such as CCK, to increase in concentration by turning the inlet media flow rate “OFF” to the IC circulation loop and the EC circulation loop.
  • IC or EC Outlet can be used in this configuration.
  • Table 34 describes the bags of solution attached to each line when performing Feed Cells. These solutions and corresponding volumes are provided as one example of default settings that may be used.
  • step 1 shown in Table 35 may be used.
  • each day the cell culture is sampled for cell counts using the following settings:
  • a length of tubing of about six (6) inches long (1 mL) is excised.
  • the volume in this sample provides a representative cell concentration sample of the entire IC loop. This allows the user(s) to monitor the cells throughout the duration of culturing.
  • This part of the example protocol is performed to continuously add a low flow rate to the IC circulation loop and/or the EC circulation loop once the cell culture conditions have reached a minimum tolerance glucose concentration or maximum tolerance lactate concentration. This may occur earlier or later than day 4, in embodiments.
  • Table 37 describes the bags of solution attached to each line when performing Feed Cells. These solutions and corresponding volumes are provided as one example of default settings that may be used.
  • step 1 shown in Table 38 may be used.
  • each day the cell culture is sampled for cell counts (see Table 36, for example).
  • This part of the example protocol is performed to transfer cells in suspension from the IC circulation loop, including cells in the bioreactor, to the harvest bag.
  • Table 39 describes the bags of solution attached to each line when performing Harvest Cells. These solutions and corresponding volumes are provided as one example of default settings that may be used.
  • the values for Harvest Cells shown in Table 40 may be used.
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US11795432B2 (en) * 2014-03-25 2023-10-24 Terumo Bct, Inc. Passive replacement of media
US20190382709A1 (en) * 2016-05-05 2019-12-19 Terumo Bct, Inc. Automated Production and Collection

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